Pharmaceuticals derived from animals continuously make a major contribution to health in terms of prevention and treatment of many diseases [
1,
2,
3,
4]. Animal venoms and their components, such as those from snakes, scorpions, spiders, bees, wasps, snails, toads, frogs, lizards and sea anemones have long been used in scientific research and are the basis of many products and drugs that are of great use in medicine today [
5,
6,
7,
8,
9,
10,
11,
12]. Crude venoms are complex bioactive chemicals rich in proteins and peptides with diverse pharmacological actions that are often protease-resistant due to their disulphide-rich structures. For example, in spider venoms the disulphide bridges form “cysteine knots”, where three bridges stabilize antiparallel beta sheets, drastically increasing the stability of the protein [
13]. The components of venoms are specific, stable, potent and have the ability to modify molecular targets, thus making good therapeutic candidates. Animal venoms have been used as a traditional medicine to treat a variety of conditions, including arthritis, rheumatism and chronic pain as well as autoimmune, cardiovascular and skin diseases [
3,
6,
14,
15,
16,
17]. Moreover, one of the most promising fields in venom research from the therapeutic aspect is their use in anticancer research. This is driven by the resistance to chemotherapeutics by cancerous cells that is making cancer treatment more complicated, hence, animal venoms have emerged as an alternative strategy for anticancer therapeutics and could also impact the costs related to cancer treatment [
15,
18,
19,
20,
21,
22,
23]. The anticancer activities of animal venoms include the inhibition of the proliferation of cancer cells, their invasion, cell cycle arrest, induction of apoptosis or necrosis and the identification of the involved signalling pathways [
14,
15,
24].
1. Coelenterata
Venomous jellyfish produce numerous problems for both tourists and fishermen in coastal areas where jellyfish are abundant. The possible genotoxic action of the venom extracted from the Mediterranean jellyfish
Pelagia noctiluca nematocysts was studied in green monkey kidney (Vero) and human colon cancer (HCT 116) cells at concentrations of 80, 160, 320 and 640 μg/mL. An alkaline comet assay indicated dose-dependent DNA damage. DNA breakage was observed in both cell cultures. It is presumed that the breaks arose due to the oxidative stress triggered by the venom, whereas later DNA lesions favoured the development of apoptosis [
53,
54]. When non-fractionated nematocysts were incubated with green monkey Vero cells, dose-dependent DNA damage was also observed with a maximum effect produced by 150,000 nematocysts per mL [
53].
2.2. Sponges (Poryphera)
Sponges possess a very potent secondary metabolism, producing numerous alkaloids, terpenoids and other biologically active compounds. These products compensate for the lack of an immune system in this taxon. Chemical compounds isolated from sponges are tested for various pharmacological effects [
55,
56]. Although mostly marine sponges are in the focus of interest, freshwater animals also synthesize numerous active compounds. The acetone extract of the freshwater sponge
Ochridaspongia rotunda manifested cytotoxic and antiradical activities; however, the extract did not induce DNA breaks [
57]. Similarly, the extract of the marine sponge
Agelas oroides produced cytotoxic effects and induced reactive oxygen species (ROS) formation and apoptosis but did not induce DNA breaks [
58]. On the contrary, aqueous and alcoholic–aqueous extracts from
Aplysina fulva, a Brazilian marine sponge, produced DNA breakage in Balb/c 3T3 mouse fibroblasts quantified by the alkaline comet assay compared with untreated cells, whereas the extent of the breakage decreased with the dilution of the extracts (1:2; 1:5; 1:10) [
59].
The genotoxic effects of individual compounds isolated from sponges also differ from the genotoxicity point of view. The terpenoid avarol from
Dysidea avara induced DNA breaks in a human cancer (HT29) cell line and Friend leukaemia cells assayed by alkaline single-cell electrophoresis [
30,
31]. The DNA damaging effect was due to the formation of hydroxyl radicals generated in the quinone/hydroquinone (avarol/avarone) cycle, as the compound does not bind DNA covalently [
30]. However, avarol appears to bind DNA non-covalently, both via intercalation and minor groove binding with subsequent DNA damage [
60].
Another sponge terpenoid, 10-acetylirciformonin B (
Figure 1), produced double-strand breaks detected both by the neutral comet assay and histone γ-H2AX expression in human leukaemia (HL 60) cells; the DNA breakage was followed by apoptosis. In cells treated with the compound at concentrations of 1.25 and 2.5 μg/mL, the mean tail moment in the neutral comet assay increased in a dose-dependent manner compared with the untreated control, and the intensity of the γ-H2AX band followed the same trend [
32]. Ingenamine G (
Figure 1), an alkaloid isolated from the Brazilian marine sponge
Pachychalina alcaloidifera (Haplosclerida), induced DNA breaks in human lymphocytes. The DNA damage was evaluated by alkaline comet assay at concentrations of 5, 10, 15 and 20 μg/mL and compared with the untreated control. It was suggested that the compound affects the construction of a mitotic fuse [
33]. According to the results of synchrotron beam diffraction on the crystal variolin B (
Figure 1), an alkaloid with anticancer activity from the Antarctic sponge
Kirckpatrickia variolosa binds DNA via the intercalative mode [
34]. Acetylated bile acids from
Siphonochalina fortis did not induce DNA breaks in human lymphocytes [
61].
Figure 1. Some low-molecular substances from animal venoms.
2.3. Annelida
Genotoxic effects were produced by tissue extracts of marine polychaetes. DNA breaks were produced in mussel gills by a skin extract from
Hediste diversicolor and
Glycera alba jaw proboscis. It was suggested that
H. diversicolor secretes toxins via the skin for protection against predators, but
G. alba secretes toxins for predation [
62].
2.4. Mollusca
The venom of very dangerous snails of the
Conus genus is composed of hundreds of peptides called conotoxins. Some are used as analgesics, whereas other pharmacological applications are sought [
63]. Data on the possible genotoxic action of these species are scarce; however, it was shown that the NMDA receptor agonist ω-conotoxin did not produce DNA damage in the spinal cords and blood cells of Wistar rats. The alkaline comet assay demonstrated the protective effect of the CGX-1007, a recombinant analogue of ω-conotoxin, against DNA damage produced by staurospirine in primary cultures of forebrain neurons [
35].
2.5. Arachnida
This taxon is represented by numerous venomous animals, scorpions and spiders. Venoms of
Arachnida are complex mixtures of proteins and low-molecular organic compounds and are mainly neurotoxic [
9,
64].
2.5.1. Scorpions
The venom of a Brazilian scorpion
Tityus stigmurus produces DNA damage in the blood and testicular cells of Swiss mice, probably by triggering oxidative stress [
65], consequently resulting in reproductive disorders in animals and humans. The animals received a single dose of the venom’s 1/2 LD
50 (0.387 mg/kg), the effects were monitored at different time points up to 48 h, six animals per point. Alkaline comet assays indicated accumulation of DNA breaks, peaking at 2 h for both organs [
65]. Similarly, the bolus injection of the venom of the most dangerous Brazilian yellow scorpion (
Tityus serrulatus) at a dose corresponding to 1/2 LD
50 (0.90 mg/kg) produced DNA damage in several organs (hippocampus, cortex, striatum, blood, heart, lung, liver and kidney) of Swiss mice. DNA damage was monitored by the alkaline comet assay, the effect appeared one hour after injection and remained at the same level for 12 h, 8–10 animals were taken for a time point [
66]. The venom of the Indian black scorpion (
Heterometrus bengalensis Koch) produced DNA damage in human leukemic cell lines (U937 and K562) leading to apoptosis. Cells were treated with the venom at an IC
50 concentration (41.5 μg/mL for U937 and 88.3 μg/mL for K562) and the alkaline comet assay was performed 48 h later. Compared with the control cells, the comet tail length increased in the treated U937 cells by 26.4% and to 80.7% in K562 cells [
67].
2.5.2. Araneae
Despite a great number of venomous spider species, data about the genotoxic effects of these venoms are scarce. There is a report on the genotoxic effects of sphingomyelinase D isolated from venoms of the
Loxosceles genus spiders on human (HaCaT) keratinocytes. The enzyme binds the plasmatic membrane of the cells and triggers an increase in the intracellular superoxide level, the radical that produces DNA damage [
68]. The venom of the
Phoneutria nigriventer spider manifests analgesic effects. It was shown that the Phα1β peptide isolated from this venom induced DNA damage in the spinal cord cells of Wistar rats but not in white blood cells. The Wistar rats were administered native Phα1β toxin intrathecally at 500 pmol/site and its recombinant analogue at 200, 500 and 1000 pmol/site. Positive control animals received hydrogen peroxide, and negative control animals received phosphate-buffered saline; there were five animals per group. The increased micronucleus frequency in bone marrow cells suggested mutagenic effects [
36].
2.6. Insecta
Hymenoptera venoms consist of a complex mixture of chemically or pharmacologically bioactive components including phospholipases, hyaluronidases and mastoparans [
69]. Mastoparans, a group of alpha-helical peptides, appear to be the most promising for pharmacological activities [
37] since they are able to penetrate into cells and bind DNA [
70]. Melectin, an antimicrobial peptide from the venom of the cleptoparasitic bee
Melecta albifrons, does not exhibit sequence homology with other wasp venom peptides. Melectin manifests antitumour activity, penetrates membranes and binds DNA [
38]. The venom of a parasitoid wasp,
Pteromalus puparum, contains a peptide with endonuclease activity (PpENVP), which inhibits gene expression in transfected cells relying on two activation sites [
71].
It is believed that
Hymenoptera contain substances able to decrease the genotoxic or mutagenic action of other compounds. A protective effect of the Egyptian honeybee (
Apis mellifera lamarckii) venom against the damaging action of propionic acid (250 mg/kg 3 days) was observed in the neurons of Sprague–Dawley rat pups. DNA damage was assayed by single-cell electrophoresis in alkaline conditions, 0.5 mg/kg of the venom was administered for 4 weeks, and there were 10 pups in a group. Both DNA damage and markers of oxidative stress were decreased [
72]. Moreover, the radioprotective effects of honeybee venom (
Apis mellifera) against 915 MHz microwave-radiation-induced DNA damage in Wistar rat lymphocytes was observed
in vitro [
73]. On the other hand, the
A. mellifera venom even at low concentrations (0.1, 0.05 and 0.01μg/mL) did not protect HepG2 cells against the genotoxic action of methyl methanesulfonate [
74]. In a similar study conducted with venom from the wasp
Polybia paulista, it was shown that higher concentrations of the venom (10, 5 and 1 μg/mL) were genotoxic
per se, whereas lower concentrations (1 ng/mL, 100 and 10 pg/mL) were not genotoxic; neither displayed a genoprotective effect. The genotoxic and mutagenic activity of the venom of
P. paulista could have been caused by phospholipase, mastoparan and hyaluronidase, as these enzymes disrupt the cell membrane and thereby interact with the genetic material of the cells or even facilitate the entrance of other compounds of the venom that can act on the DNA. Furthermore, venom substances are able to trigger inflammatory process and generate ROS that can interact with the DNA [
75].
Bee venom (
A. mellifera) and its mayor constituent melittin (
Figure 1) are cytotoxic towards human peripheral blood cells and are able to induce morphological changes in the cell membrane, granulation and lysis of the cells. Moreover, they showed increased DNA damage including oxidative DNA damage as well as increased formation of other markers of genomic instability. This genotoxicity coincides with the increased formation of ROS, reduction of glutathione and increased lipid peroxidation as well as phospholipase C activity, indicating the induction of oxidative stress. Melittin itself is also capable of modulating the gene expression patterns of genes involved in the DNA damage response, oxidative stress and apoptosis [
25,
26,
27,
39,
40,
41].